Skeletal development and homeostasis depend on a higher-order network that ensures efficient balance between bone formation by osteoblasts and resorption by osteoclasts. Perturbations of this network are often associated with skeletal disorders, with the most severe being osteoporosis, with inherent risk of fracture and associated morbidity and mortality. A master regulator of bone remodeling is the canonical Wnt signaling pathway, which exerts a tight control over several aspects of osteoblast differentiation. The homeodomain protein TGIF functions as a transcriptional corepressor in the transforming growth factor beta (TGF-) signaling pathway, which is known to restrict bone formation. To advance our understanding of how TGIF's physiological functions and regulation are controlled, we have screened for TGIF-binding proteins using the yeast two-hybrid assay. A previously unidentified TGIF binding protein that attracted our attention is Axin2, which functions as a negative regulator of Wnt signaling through its potential to mediate formation of the destructosome, a signaling module where the protein kinase GSK3- marks -Catenin for ubiquitin-dependent degradation. In subsequent gain- and loss-of-function studies, TGIF was found to promote Wnt-induced transcriptional responses. Our preliminary investigations also suggest that TGIF is essential to the ability of Wnt signaling to induce osteoblast differentiation. Strikingly, we found that TGIF can regulate bone formation by a mechanism independent of its ability to suppress TGF- signaling, revealing that an alternative physiological function of TGIF is to ensure effective osteoblast cell fate determination in response to Wnt. Consistent with its role in osteoblast commitment, ablation of TGIF in mice impaired osteoblast maturation and decreased bone mass. Taken together, these findings led us to propose a working hypothesis in which TGIF may contribute to the regulation of canonical Wnt signaling in bone, imposing a stringent control over the destructosome scaffolding complex. Accordingly, the specific aims of this research proposal are: Aim 1: Further delineate the molecular mechanisms by which TGIF affects Wnt-mediated osteoblast differentiation and bone formation, with particular emphasis on its possible role in the assembly/disassembly of the destructosome, and thereby -Catenin stability. Aim 2: Analyze the effects of full and osteoblast-targeted deletion of TGIF on bone homeostasis and the ability of Wnt to induce osteoblast differentiation and bone formation in vitro and in vivo. Functional characterization of TGIF as a bona fide contributor to the regulation of Wnt signaling and bone formation, will strengthen our knowledge of key physiological processes that maintain bone homeostasis, ultimately evolving into new concepts in the design and implementation of safe anabolic therapeutic drugs against osteoporosis and other low bone mass syndromes. PUBLIC HEALTH RELEVANCE: Wnt signaling fosters osteoblast proliferation and differentiation, and perturbations of this signaling pathway affect bone homeostasis, resulting in bone disorders in human. We discovered that a physiological function of the homeodomain protein TGIF is to ensure proper osteoblast cell fate determination in response to Wnt signaling. We believe that functional characterization of his newly discovered regulator of bone formation will open a new field of investigation both in terms of understanding the mechanisms that regulate osteoblasts and their bone matrix-secreting activity and in terms of drug discovery with the hope to improve the treatment of osteoporosis and other low bone mass syndromes.